Production of bifunctional proteins by Aspergillus awamori: Llama variable heavy chain antibody fragment (V HH) R9 coupled to Arthromyces ramosus peroxidase (ARP)

• Published in: Journal of biotechnology Vol. 120; no. 4; pp. 347 - 359
• Main Authors: Joosten, Vivi, Roelofs, Marc S., van den Dries, Niels, Goosen, Theo, Verrips, C. Theo, van den Hondel, Cees A.M.J.J., Lokman, B. Christien
• Format: Journal Article
• Language: English
• Published: Lausanne Elsevier B.V 06.12.2005; Amsterdam Elsevier; New York, NY
• Subjects: Animals; Antibodies, Monoclonal - biosynthesis; Antibodies, Monoclonal - genetics; Antibodies, Monoclonal - immunology; Arthromyces ramosus; Arthromyces ramosus peroxidase (ARP); Aspergillus; Aspergillus awamori; Aspergillus awamori ;Heterologous protein production; Biological and medical sciences; Biotechnology; Camelids, New World - genetics; Camelids, New World - immunology; domain; endoplasmic-reticulum; filamentous fungi; Fundamental and applied biological sciences. Psychology; Fungal Proteins - biosynthesis; Fungal Proteins - genetics; Fungi, Unclassified - enzymology; Fungi, Unclassified - genetics; fusion; Fusion proteins; gene; heterologous expression; Immunoglobulin Heavy Chains - biosynthesis; Immunoglobulin Heavy Chains - genetics; Immunoglobulin Heavy Chains - immunology; Immunoglobulin Variable Region - biosynthesis; Immunoglobulin Variable Region - genetics; Immunoglobulin Variable Region - immunology; light-chains; Llama variable heavy chain antibody fragments (V HHs); Magic Bullets; Peroxidase - biosynthesis; Peroxidase - genetics; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - immunology; saccharomyces-cerevisiae; secretion; thaumatin production; Arthromyces ramosus peroxidase (ARP); Magic Bullets; Fusion proteins; Aspergillus awamori ;Heterologous protein production; Llama variable heavy chain antibody fragments (V HHs); Heavy peptide chain; Enzyme; Antibody; Fragment; Fungi; Peroxidases; Production; Peroxidase; Fungi Imperfecti; Oxidoreductases; Recombinant protein; Fusion protein; Llama variable heavy chain antibody fragments (VHHs); Thallophyta; Aspergillus awamori
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2005.06.034

The Arthromyces ramosus peroxidase gene ( arp) was genetically fused to either the 5′- or 3′-terminal ends of the gene encoding llama variable heavy chain antibody fragment V HH R9, resulting in the fusion expression cassettes ARP-R9 or R9-ARP. Aspergillus awamori transformants were obtained which produced up to 30 mg l −1 fusion protein in the culture medium. Both fusion proteins showed peroxidase activity in an ABTS activity test. Considerable amounts of fusion protein were detected intracellularly, suggesting that the fungus encounters problems in secreting these kind of proteins. ELISA experiments showed that ARP-R9 was less able to bind its antigen, the azo-dye RR6, as compared to R9-ARP. Furthermore, in contrast to R9-ARP, ARP-R9 bound to RR6 did not show peroxidase activity anymore. These results indicate that fusion of ARP to the C-terminus of the antibody fragment V HH R9 (R9-ARP) is the preferred orientation.